The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3, referred to as A3) proteins are cellular cytidine deaminases that potently restrict retrovirus replication. However, HIV-1 viral infectivity factor (Vif) counteracts the antiviral activity of most A3 proteins by targeting them for proteasomal degradation. To date, the structure of an A3 protein containing a Vif-binding interface has not been solved. Here, we report a high-resolution crystal structure of APOBEC3C and identify the HIV-1 Vif-interaction interface. Extensive structure-guided mutagenesis revealed the role of a shallow cavity composed of hydrophobic or negatively charged residues between the α2 and α3 helices. This region is distant from the DPD motif (residues 128-130) of APOBEC3G that participates in HIV-1 Vif interaction. These findings provide insight into Vif-A3 interactions and could lead to the development of new pharmacologic anti-HIV-1 compounds.

New methods for the synthesis of artificial metalloenzymes are important for the construction of novel biocatalysts and biomaterials. Recently, we reported new methodology for the synthesis of artificial metalloenzymes by reconstituting apo-myoglobin with metal complexes (Ohashi, M. et al., Angew Chem., Int. Ed. 2003, 42, 1005-1008). However, it has been difficult to improve their reactivity, since their crystal structures were not available. In this article, we report the crystal structures of M(III)(Schiff base).apo-A71GMbs (M = Cr and Mn). The structures suggest that the position of the metal complex in apo-Mb is regulated by (i) noncovalent interaction between the ligand and surrounding peptides and (ii) the ligation of the metal ion to proximal histidine (His93). In addition, it is proposed that specific interactions of Ile107 with 3- and 3'-substituent groups on the salen ligand control the location of the Schiff base ligand in the active site. On the basis of these results, we have successfully controlled the enantioselectivity in the sulfoxidation of thioanisole by changing the size of substituents at the 3 and 3' positions. This is the first example of an enantioselective enzymatic reaction regulated by the design of metal complex in the protein active site.

Eukaryotic 20S proteasomes are composed of two alpha-rings and two beta-rings, which form an alphabetabetaalpha stacked structure. Here we describe a proteasome-specific chaperone complex, designated Dmp1-Dmp2, in budding yeast. Dmp1-Dmp2 directly bound to the alpha5 subunit to facilitate alpha-ring formation. In Deltadmp1 cells, alpha-rings lacking alpha4 and decreased formation of 20S proteasomes were observed. Dmp1-Dmp2 interacted with proteasome precursors early during proteasome assembly and dissociated from the precursors before the formation of half-proteasomes. Notably, the crystallographic structures of Dmp1 and Dmp2 closely resemble that of PAC3-a mammalian proteasome-assembling chaperone; nonetheless, neither Dmp1 nor Dmp2 showed obvious sequence similarity to PAC3. The structure of the Dmp1-Dmp2-alpha5 complex reveals how this chaperone functions in proteasome assembly and why it dissociates from proteasome precursors before the beta-rings are assembled.

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The ubiquitin ligase complex SCF(Fbs1), which contributes to the ubiquitination of glycoproteins, is involved in the endoplasmic reticulum-associated degradation pathway. In SCF ubiquitin ligases, a diverse array of F-box proteins confers substrate specificity. Fbs1/Fbx2, a member of the F-box protein family, recognizes high-mannose oligosaccharides. To elucidate the structural basis of SCF(Fbs1) function, we determined the crystal structures of the Skp1-Fbs1 complex and the sugar-binding domain (SBD) of the Fbs1-glycoprotein complex. The mechanistic model indicated by the structures appears to be well conserved among the SCF ubiquitin ligases. The structure of the SBD-glycoprotein complex indicates that the SBD primarily recognizes Man(3)GlcNAc(2), thereby explaining the broad activity of the enzyme against various glycoproteins. Comparison of two crystal structures of the Skp1-Fbs1 complex revealed the relative motion of a linker segment between the F-box and the SBD domains, which might underlie the ability of the complex to recognize different acceptor lysine residues for ubiquitination.

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Apo-myoglobin (apo-Mb) and apo-A71GMb were successfully reconstituted with FeIII(salophen) (1) (salophen = N,N'-bis(salicylidene)-1,2-phenilenediamine), Fe(III)(3,3'-Me2-salophen) (2), and FeIII(5,5'-t-Bu2-salophen) (3). The crystal structure of 2.apo-A71GMb shows the tight binding of the complex in the Mb cavity, while in wild-type apo-Mb it is highly disordered due to the steric repulsion with Ala71. Furthermore, the structure of 2.apo-A71GMb suggests a possible accommodation of a small substrate in the cavity. In fact, the cyanide association rate constant of 2.apo-A71GMb is 216-fold larger compared to that of 2.apo-Mb. These results provide us principles for the noncovalent fixation of synthetic metal cofactors at the desired positions in protein matrixes.